HIV-1 packs in PACSIN2 for cell-to-cell spread

The assembly and release of retroviral particles is driven primarily by the Gag polyprotein precursor, which contains several functional domains: matrix, capsid, and nucleocapsid. The matrix domain directs Gag to the plasma membrane, capsid plays a key role in Gag multimerization during assembly, and nucleocapsid binds to the viral genomic RNA, recruiting it into the assembling virus particle. In addition to matrix, capsid, and nucleocapsid domains, retroviral Gag precursors contain a variety of smaller peptides, some of which harbor short motifs known as “late” domains that promote virus particle release from the infected cell (1, 2). In PNAS, Popov et al. (3) show that ubiquitin modification of HIV-1 Gag recruits the cellular protein PACSIN2 to sites of assembly to promote virus spread at points of cell-to-cell contact.

Cells encode a complex machinery that catalyzes membrane scission events that are oriented away from the cytoplasm; these scission events are essential for a variety of key cellular processes, including the abscission step of cytokinesis, and the generation of intralumenal vesicles that bud into late endosomes to form multivesicular bodies. At the core of this membrane scission machinery are the endosomal sorting complexes required for transport (ESCRTs)—ESCRT-0, -I, -II, and -III—and ESCRT-associated factors, such as ALIX and the AAA ATPase Vps4. Retroviral late domains promote virus particle pinching-off from the plasma membrane by recruiting components of this machinery (2). Retroviruses encode three distinct late-domain sequences: Pro-Thr-Ala-Pro (PTAP), Tyr-Pro-Xn-Leu (YPX_nL, where X_n represents 1–4 variable amino acids), and Pro-Pro-X-Tyr (PPXY). These three late domains bind directly to the ESCRT-I component Tsg101, ALIX, and NEDD4-family ubiquitin ligases, respectively. Budding of the paramyxovirus PIV5 was shown to be dependent on the host protein angiomotin-like 1 (AmotL1) (4) and the related protein angiomotin has been implicated in HIV-1 release (5). The motin family of proteins contain BAR domains (for Bin, Amphiphysin, and Rvs), which are coiled–coiled motifs that sense or induce membrane curvature (6).

Ubiquitin modification plays a key role in ESCRT function, and several components of this apparatus contain ubiquitin-binding domains (7). For example, the sorting of cargo proteins into multivesicular bodies often requires the ubiquitination of the cargo. Although many retroviral Gag proteins, including that of HIV-1, are ubiquitinated (8), the precise role of this modification in retroviral budding and virus replication is unclear. As mentioned above, PPXY-type late domains bind directly to NEDD4-family ubiquitin ligases. Further supporting a role for Gag ubiquitination in retroviral budding, appending a ubiquitin to the C terminus of a retroviral Gag protein can rescue budding defects imposed by late-domain mutation (9), and fusion of a deubiquitinating enzyme to the C terminus of HIV-1 Gag suppresses particle budding (10). Finally, overexpression of the NEDD4-2s/NEDD4L ubiquitin ligase rescues the budding defect imposed by mutation of the HIV-1 PTAP motif (11, 12).

Once a virus particle buds from the cell, the viral protease cleaves at a number of sites within the Gag polyprotein precursor, triggering a morphological transformation of the newly released virion known as maturation. The mature, infectious particle can then be transmitted to target cells by two general modes: as cell-free virus or by transfer from infected cell to uninfected cell across a point of close cell-to-cell contact known as a virological synapse (VS) (13). Formation of the VS, a structure that is analogous in certain respects to the immunological synapse (14), is promoted by adhesion molecules, actin, and binding of the viral envelope glycoprotein to its receptor (15–18). Although the role of cell-to-cell transfer in retroviral spread in vivo remains a matter of debate, many studies have demonstrated that, in cell culture, this mode of viral transfer is substantially more efficient than cell-free infection (19–21).

In PNAS, Popov et al. (3) examine the role of cellular proteins that are recruited by the PPXY late domain of the avian retrovirus Rous sarcoma virus. Using a proteomics approach, the authors identified host proteins that were selectively incorporated into virus-like particles (VLPs) by binding to PPXY. As expected, several components of the ubiquitin ligase pathway and ESCRT machinery were identified. The screen also identified the BAR domain-containing factor protein kinase C and casein kinase substrate in neurons 2 (PACSIN2) and its interaction partners, the Eps15 homology domain (EHD)-containing proteins, EHD1 and EHD4. Because BAR domain-containing proteins have been implicated in HIV-1 assembly, here Popov et al. focus on PACSIN2 recruitment by HIV-1 Gag. Despite lacking a PPXY late domain, HIV-1 Gag p6 also directed the incorporation of PACSIN2 into VLPs in a manner that correlated with the extent of Gag ubiquitination. Additionally, overexpressing the NEDD4-2s ubiquitin ligase enhanced both the levels of Gag ubiquitination and the incorporation of PACSIN2 into VLPs, supporting the hypothesis that Gag-ubiquitin conjugates recruit PACSIN2.

To investigate a potential role for PACSIN2 in HIV-1 replication, Popov et al. (3) knocked down PACSIN2 expression in HIV-1 susceptible T cell lines and primary T cells. They observed that PACSIN2 depletion impaired the ability of the cells to support a spreading HIV-1 infection, a defect that could be rescued by reintroducing PACSIN2. The Src-homology 3 (SH3) domain of PACSIN2, which is known to interact with actin remodeling proteins (22), was required for the rescue, suggesting that PACSIN2 may modulate the actin environment in the vicinity of virus assembly sites. Interestingly, PACSIN2 depletion had no effect on either the efficiency of HIV-1 particle production or the infectivity of HIV-1 virions. Instead, PACSIN2 depletion in virus-producing cells markedly impaired HIV-1 cell-to-cell transmission. These results support a model whereby HIV-1 Gag-containing ubiquitin modifications recruits PACSIN2 to promote cell-to-cell transmission at the VS (Fig. 1).

In PNAS, Popov et al. show that ubiquitin modification of HIV-1 Gag recruits the cellular protein PACSIN2 to sites of assembly to promote virus spread at points of cell-to-cell contact.

Fig. 1.

Proposed model for the recruitment of PACSIN2 to HIV-1 assembly sites by ubiquitinated Gag. HIV-1 Gag—composed of matrix (MA), capsid (CA), nucleocapsid (NC), and p6 domains—orchestrates the recruitment of host cellular proteins to sites of virus assembly at the plasma membrane. Virus particles assembled in the producer cell (donor, Left) can be transferred to an uninfected cell (target, Right) by either a cell-free or cell-associated mechanism, both of which require interactions between the viral envelope (Env) glycoprotein and the CD4 receptor molecule. Ubiquitinated Gag (Gag-Ub) recruits PACSIN2 (structure based on PDB ID code 3HAJ) to sites of assembly and is incorporated into viral particles; PACSIN2 specifically enhances the efficiency of HIV-1 spread by cell-to-cell transfer across a VS but is not required for cell-free infection. Cellular adhesion molecules play a role in VS formation; PACSIN2 is proposed to bridge the membrane and the underlying actin cytoskeleton.

The findings of Popov et al. (3) are significant in several respects. First, they identify a host factor, PACSIN2, which appears to play a vital role in mediating HIV-1 cell-to-cell transfer. Although it has long been known that actin reorganizes at the VS (17), the new data of Popov et al. (3) suggest that PACSIN2 could bridge Gag and actin to facilitate this process. Second, the results identify a heretofore unappreciated function for Gag-ubiquitin conjugates in recruiting cellular machinery that enhances HIV-1 replication. Third, the Popov et al. study underscores the importance of cell-to-cell transfer in HIV-1 spread, at least in vitro; although PACSIN2 depletion has no measurable effect on virus particle assembly and release or virion infectivity, it effectively shuts down the establishment of a spreading infection.

Interesting questions remain: where in Gag are the key ubiquitin molecules that mediate PACSIN2 recruitment? Which cellular ubiquitin ligase is responsible for HIV-1 Gag ubiquitination under physiological conditions? What role does PACSIN2 play in viral transmission across the VS? The observation that PACSIN2 is not required for virus assembly and budding argues against a role in helping Gag to bend membrane; rather, PACSIN2’s reported function in inducing the formation of filopodia (23) suggests that the BAR domain-containing protein may promote the formation of curved cellular structures that serve as hot spots for VS formation and viral transfer. Finally, because many retroviruses rely heavily on cell-to-cell transfer, and many retroviral Gag proteins are modified with ubiquitin, it will be of interest to determine whether retroviruses other than HIV-1 recruit PACSIN2, or potentially other BAR domain proteins, to facilitate their transmission. Ultimately, a key question to address will be to what extent is cell-to-cell transfer, and PACSIN2’s role in this process, required for retroviral spread in vivo.